Synthetic peptides, conjugation reagents and methods

ABSTRACT

The invention provides methods and compositions useful for making synthetic peptide conjugates. In one embodiment, the invention provides compositions comprising the structure:  
                 
wherein R is selected from lower substituted or unsubstituted alkyl, O, NH and S and P is an amine protection group. In more particular embodiments, the compositions comprise α-amine protected 4,5-dehydroleucine or α-amine protected (2S)-aminolevulinic acid and/or P is F-moc. These compounds may be incorporated into peptides, for example, peptides comprising a substituted or unsubstituted (2S)-aminolevulinic acid residue, such as (2S)-aminolevulinic acid residue is substituted with an O- or N-linked glycoconjugate, or a detectable label.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of Ser. No. 10/268,813,filed Oct. 10, 2002, now U.S. Pat. No. 6,939,945, which is a divisionalapplication of Ser. No. 09/405,516, filed Sep. 23, 1999, now U.S. Pat.No. 6,465,612, which claims priority to application Ser. No. 60/101,494,filed Sep. 23, 1998, all of which are incorporated herein by reference.

U.S. GOVERNMENT RIGHTS

The research carried out in the subject application was supported inpart by grants from the National Science Foundation (No. CHE-9734430).The government may have rights in this invention.

INTRODUCTION

1. Field of the Invention

The invention relates to methods and reagents used to make syntheticpeptide conjugates.

2. Background of the Invention

Glycoprotein pharmaceuticals are major targets of the biotechnologyindustry and include such widely used therapeutic agents as tissueplasminogen activator (TPA), erythropoietin (EPO) and monoclonalantibodies. Glycosylation presents special challenges in drug discoveryand development, largely due to the heterogeneity of oligosaccharidestructures obtained from recombinant expression in eukaryotic celllines. The presence of heterogeneous glycoforms convolutes thecharacterization of the glycoprotein's structure and biologicalactivity, which hinders clinical evaluation and approval. New strategiesfor their production which control oligosaccharide structure anduniformity would facilitate the development of glycoproteinpharmaceutical agents.

We have developed novel methods for the synthesis of glycopeptides basedon the highly selective reaction of nucleophilic carbohydtratederivatives with ketone-containing peptides. Peptides bearing unnaturalketone side chains can be generated using N-protectd (2S)-aminolevulinicacid by standard solid-phase peptide synthesis (SPPS). Oligosaccharidesfunctionalized at their reducing termini with aminooxy, hydrazide orthiosemicarbazide groups can be coupled to keto-peptides in aqueoussolvent without need for protecting groups or auxiliary couplingreagents. These methods can be used to prepare glycopeptides oftherapeutic interest.

SUMMARY OF THE INVENTION

The invention provides methods and compositions useful for makingsynthetic peptide conjugates. In one embodiment, the invention providescompositions comprising the structure:

wherein R is selected from lower substituted or unsubstituted alkyl, O,NH and S and P is an amine protection group. In more particularembodiments, the compositions comprise α-amine protected4,5-dehydroleucine or α-amine protected (2S)-aminolevulinic acid and/orP is F-moc. These compounds may be incorporated into a wide variety ofsynthetic molecules such as peptides. In a particular embodiment, suchpeptides comprise a substituted or unsubstituted (2S)-aminolevulinicacid residue, such as (2S)-aminolevulinic acid residue is substitutedwith an O- or N-linked glycoconjugate, or a detectable label. Thesepeptides may be synthesized in vitro or in vivo and may be incorporatedinto cells or cellular structures, e.g. by direct feeding to the cells.Feeding ketone-bearing precursors to cells for incorporation intocellular structures was well-known in the art as of our filing date;see, e.g. Mahal et al., May 1997, Science 276, 1125-28.

The invention also provides methods for conjugating a molecule to acomposition comprising a (2S)-aminolevulinic acid residue comprising thestep of reacting the molecule with the residue under conditions wherebythe molecule is covalently conjugated to residue. In particularembodiments, the composition comprises a synthetic peptide comprisingthe (2S)-aminolevulinic acid residue or the molecule is a glycoconjugateor comprises a detectable label.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

The following descriptions of particular embodiments are offered by wayof illustration and not by way of limitation. Unless contraindicated ornoted otherwise, in these descriptions and throughout thisspecification, the terms “a” and “an” mean one or more, the term “or”means and/or.

EXAMPLE I

Direct incorporation of unprotected ketone groups into peptides duringsolid phase synthesis: application of the one-step modification ofpeptides with two different biophysical probes for FRET.

Abstract: An amino acid bearing an unprotected ketone group,(2S)-aminolevulinic acid, was incorporated into a synthetic peptideusing standard Fmoc-based solid-phase methods. The ketone group remainedunharmed during the synthesis and provided a uniquely reactivefunctional group for covalent modification of the peptide. The ketoneand the sulfhydryl group of a cysteine residue elsewhere in the peptidewere reacted simultaneously with two different biophysical probes,enabling the site-specific installation of a donor and acceptor pair forFRET in one step without the need for differential side chainprotection.

The fluoresence resonance energy transfer (FRET) technique has foundwidespread use in the study of protein structure and dynamics.¹ Thetechnique requires the installation of two biophysical probes, a donorand an acceptor, at defined locations within the peptide of interest. Anumber of methods are now available for the conjugation of smallmolecule probes to peptides and proteins. The majority of these methodsare based on coupling electrophilic functional groups, such asα-iodoacetamides² and N-hydroxysuccinimido esters,³ with exposedcysteine and lysine residues, respectively. However, in order achievesite-specific modification of a peptide in the presence of multiplecopies of these nucleophilic residues, differential side chainprotection must be employed during the synthesis. To avoid the need forextra protecting group manipulations during the conjugation of asynthetic peptide with two different probes, several groups haveexploited selective N-terminal reactions,^(1f) or incorporatednon-native amino acids with suitable photophysical properties (e.g.,p-nitrotyrosine) in place of their native counterparts.⁴ Theseapproaches are somewhat limited with regard to the site of residence andstructure of the probe.

In recent years, the adornment of synthetic peptides with a uniquelyreactive electrophile has become an increasingly popular tactic forsite-specific modification. For example, ketone or aldehyde groups canbe installed in synthetic peptides by conjugating levulinic acid(4-oxopentanoic acid) or masked aldehydes to the ε-amino groups oflysine side chains or to the N-terminus of the peptide.⁵ Alternatively,aldehyde groups can be generated by the selective oxidation ofN-terminal serine residues with periodate.⁶ The ketones or aldehydesform stable covalent adducts with a complementary nucleophile, typicallyan aminooxy, hydrazide or thiosemicarbazide group, in aqueous milieu inthe absence of side chain protecting groups. Still, these methods fordecorating peptides with ketones and aldehydes require orthogonal sidechain or N-terminal protection for site-specific introduction of theelectrophile.

Here we report that (2S)-aminolevulinic acid, an amino acid bearing anunprotected ketone group, can be directly incorporated into syntheticpeptides using standard Fmoc-based solid-phase methods. The ketone groupsurvives the synthesis without undergoing any apparent degradation orunwanted side reactions. Furthermore, the ketone is chemicallyorthogonal to all natural amino acid side chain functional groups. Thus,sulfhydryl groups of cysteine residues, for example, can be modified inthe presence of the ketone and vice versa, allowing for the selectivelabeling of a peptide with two different probes in one synthetic step.The versatility of the ketone group is therefore two-fold: it can beinstalled at any site along the polypeptide backbone without need forextra protecting group steps, and it does not interfere with reactionsinvolving the nucleophilic functional groups of native amino acid sidechains.

Fmoc-protected (2S)-aminolevulinic acid (3) was synthesized in two stepsfrom commercially available 4,5-dehydroleucine⁷ (1) as depicted inScheme 1. The α-amine was first protected with an Fmoc group to affordcompound 2,⁸ which was then converted to keto-amino acid 3 by reductiveozonolysis.⁹ We incorporated compound 3 into a 19-amino acid peptide (4)on an automated peptide synthesizer using standard Fmoc-basedsolid-phase methods.¹⁰ The sequence of peptide 4 was derived from theanti-microbial glycopeptide drosocin, which is produced by insects inresponse to immune challenge.¹¹ The bacteriostatic potency of drosocinis enhanced five-fold by the presence of an O-linked glycan at Thr11, afeature that may reflect an altered conformational preference due toglycosylation. Thus, drosocin constructs labeled with biophysical probesmay be useful tools for analyzing the conformation of the unglycosylatedpeptide compared to the glycosylated version. We chose to install the(2S)-aminolevulinic acid group in place of Ile17. In addition, acysteine residue was incorporated in place of Lys2 to provide a secondorthogonal site for covalent modification.

We were concerned that the ketone group might undergo unwanted sidereactions, such as condensation with amines to form imines or enamines,or acid- or base-catalyzed aldol condensations, during the process ofFmoc-based solid-phase synthesis. However, the crude product obtainedafter synthesis and cleavage from the resin was a single peak byreversed-phase HPLC (RP-HPLC) analysis, and its identity as the desiredketo-peptide (4) was confirmed by mass spectrometry.¹² No other sideproducts were obtained, indicating that the unprotected ketone group isfully compatible with the reagents of Fmoc-based synthesis, includingDCC, piperidine and TFA. In essence, keto-amino acid 3 can be treatedsimilarly to an alanine residue.

To demonstrate the versatility of the ketone group, we modifiedketo-peptide 4 with two biophysical probes, coumarin iodoacetamide (5)and fluorescein thiosemicarbazide (6), which are commonly used as adonor and acceptor pair for FRET.^(13,14) The site-specific labeling ofpeptide 4 with these probes was achieved in one synthetic step (Scheme2). Peptide 4 (5 mg) was incubated with 1.2 equivalents each of 5 and 6in 900 μL DMF and 100 μL of 1.0 M sodium phosphate buffer, pH 7.0. Theligation reaction was complete after 24 hours and the fluorescentlylabeled peptide (7) was isolated by RP-HPLC and its identity confirmedby mass spectrometry.¹⁵ This general strategy for attaching a pair ofsmall molecules to synthetic peptides is applicable to a wide range oftargets.

In summary, N-Fmoc-(2S)-aminolevulinic acid (3) can be incorporated intosynthetic peptides in an unprotected form to provide a convenientelectrophilic functional group, the ketone, for site-specificconjugation. The chemical orthogonality of the ketone to the sulfhydrylgroup of cysteine side chains allows for the selective modification ofpeptides with two different probes and facilitates the synthesis ofconstructs for FRET experiments. This method is therefore a usefulcomplement to traditional protein modification techniques.

REFERENCES AND NOTES

1. (a) Stryer, L. Ann. Rev. Biochem. 1978, 47, 819-846. (b) Lee, J. A.;Fortes, P. A. G. Biochemistry 1985, 24, 322-330. (c) Taniguchi, K.;Mardh, S. J. Biol. Chem. 1993, 268a, 15588-15594. (d) Wu, P.; Brand, L.Anal. Biochem. 1994, 218, 1-13. (e) Miki, M.; Kouyama, T. Biochemistry1994, 33, 10171-10177. (f) Imperiali, B.; Rickert, K. W. Proc. Natl.Acad. Sci. 1995, 92, 97-101. (g) Xing, J.; Cheung, H. C. Biochemistry1995, 34, 6475-6487. (h) Mehta, S.; Meldal, M.; Ferro, V.; Duus, J. O.;Bock, K. J. Chem. Soc., Perkins Trans. 1 1997, 1365-1374. (i) Slevin, P.R. Meth. Enzymol. 1995, 246, 300-334.

2. (a) Sharma, J.; Luduena, R. F.; J. Protein Chem. 1994, 13, 165-176.(b) Wong, S. Y. C.; Guile, G.; Dwek, R.; Arsequell, G. Biochem. J. 1994,300, 843-850. (c) Esmann, M.; Sar, P. C.; Hideg, K. Marsh, D. Anal.Biochem. 1993, 213, 336-348.

3. (a) Bradbume, J. A.; Godfrey, P.; Choi, J. H.; Mathis, J. N. Appl.Environ. Microbiol, 1993, 59, 663-668. (b) Fan, J. G.; Pope, L. E.;Vitols, K. S.; Huennekens, F. M. Biochemistry 1991, 30, 4573-4580.

4. Meldal, M.; Breddam, K. Anal. Biochem. 1991, 195, 141-147.

5. (a) Canne, L. E.; Ferre-D'Amare, A. R.; Burley, S. K.; Kent, S. B. H.J. Am. Chem. Soc. 1995, 117, 2998-3007. (b) Shao, J.; Tam, J. P. J. Am.Chem. Soc. 1995, 117, 3893-3899.

6. (a) Geoghegan, K. F.; Stroh, J. G. Bioconjugate Chem. 1992, 3,138-146. (b) Rose, K. J. Am. Chem. Soc. 1994, 116, 30-33. (c) Rose, K.;Zeng, W.; Regamey, P. -O.; Chemushevich, I. V.; Standing, K. G.;Gaertner, H. F. Bioconjugate Chem. 1996, 7, 552-556.

7. 4,5-Dehydroleucine (1) was purchased from Bachem.

8. Hardy, P. M.; Sheppard, P. W. J. Chem. Soc., Perkin Trans. 1 1983,723-729.

9. Preparation of N-Fmoc-(2S)-aminolevulinic acid (3): A solution ofFmoc-4,5-dehydroleucine (2)⁸ (1.1 g, 3.1 mmol) in 9:1 CH₂Cl₂/methanol(15 mL) was cooled to −78° C. and purged with N₂ for 10 min. A stream ofozone was passed through the solution until a blue color persisted. Thereaction mixture was then purged with N₂ (ca. 10 min) until the solutionwas no longer blue in color. Dimethylsulfide (0.58 mL, 7.9 mmol) wasadded and the solution was warmed to rt overnight. Excessdimethylsulfide and solvent were removed in vacuo and the resultingyellow syrup was purified by silica gel chromatography (10:1CHCl₃/methanol, 0.1% AcOH) and crystallized from CH₂Cl₂/hexanes toafford 0.89 g (81%) of compound 3 as a white solid: mp 136-138_C; ¹H NMR(300 MHz, CDCl₃): δ 7.76 (d, 2 H, J=7.4), 7.58 (d, 2 H, J=5.1), 7.39 (t,2 H, J=7.0), 7.30 (dt, 2H, J=6.2, 1.2), 5.85 (d, 1 H, J=7.8, N—H), 4.60(m, 1H), 4.43-4.34 (m, 1H), 4.22 (t, 1 H, J=6.9), 3.25 (dd, 1H, J=18.3,4.0 ), 2.99 (dd, 1H J=15.5, 4.0), 2.19 (s, 3H, CH₃); C¹³ NMR (100 MHz,CDCl₃): δ 175.5, 156.2, 143.7, 143.6, 141.2, 127.7, 127.0, 125.0, 120.0,119.8, 67.3, 49.5, 47.0, 45.0; FAB-HRMS calcd for C₂₀H₂₀NO₅ (M+H⁺)354.1341, found 354.1334.

10. Fields, G. B.; Noble, R. L. Int. J. Peptide Protein Res. 1990, 35,161-214.

11. (a) Bulet, et al. J. Biol. Chem. 1993, 268, 14893-14897. (b) Bulet,et al. Eur. J. Biochem. 1996, 238, 64-69.

12. ES-MS: calcd for peptide 4 (M+H⁺) 2173, found m/z 2173.

13. Coumarin iodoacetamide and flourescein thiosemicarbazide werepurchased from Molecular Probes (cat. # C-404 and F-121, respectively).

14. (a) Haugland, R. Molecular Probes Handbook of Fluorescent Probes andResearch Chemicals; 6th ed. 1996. (b) Theilen, et al. Biochemistry 1984,23, 6668-6674. (c) Odom, O. W.; Deng, H. Y.; Dabbs, E. R.; Hardesty, B.Biochemistry 1984, 23, 5069-5076.

15. Reversed-phase (C₁₈) HPLC conditions: Elution gradient: CH₃CN (B) inH₂O (A), both with 0.1% TFA (10ø60% B, over 50 min). ES-MS: calcd forpeptide 7 (M+H⁺) 2939, found m/z 2939.

EXAMPLE II Synthesis of an oxime-linked neoglycopeptide withglycosylation-dependent activity similar to its native counterpart.

Abstract: Neoglycopeptides containing an oxime sugar-peptide linkage canbe generated by coupling an aminooxy sugar with a peptide bearing aketo-amino acid. The coupling reaction can be executed in aqueous milieuwithout need for protecting groups on the peptide or saccharide, orauxiliary coupling reagents. Using this method, an oxime-linked analogof an antimicrobial peptide with glycosylation-dependent function wasprepared and found to have similar bioactivity to the nativeglycopeptide. Thus, replacement of the sugar-peptide bond with anunnatural but synthetically facile linkage can produce functionalneoglycopeptides.

An appealing strategy for the assembly of glycopeptides is theconvergent coupling of an oligosaccharide with a pre-formed peptide.While this strategy has been implemented in the synthesis of N-linkedglycopeptides,¹ O-linked glycopeptides have been prohibitive due to thedifficulty in forming a glycosidic bond in the presence ofmultifunctional proteins and carbohydrates. However, if the nativeglycosidic linkage between the sugar and the peptide backbone werereplaced with an alternate non-native linkage, a convergent synthesis ofO-linked glycopeptides could be realized. To date, this strategy has notbeen widely explored, perhaps due to the suggestion that the proximalGalNAc residue of O-linked glycoproteins plays a role in modulatinglocal peptide conformation.² However, the importance of the nativesugar-peptide linkage for the bioactivity of O-linked glycopeptides,especially those with glycosylation-dependent function, has yet to beaddressed. It may be that in some cases this linkage can be substitutedwithout dramatically altering global structure or function. Thecomplexity of glycopeptide synthesis could certainly be reduced byreplacing the sugar-peptide linkage with a more facile bond.

We synthesized an O-linked glycopeptide with glycosylation-dependentactivity (1) and a neoglycopeptide analog (2) in which the sugar andpeptide are linked by an oxime (Formulae 1a). Glycopeptide 1, nameddrosocin, is an antimicrobial substance produced by insects in responseto immune challenge, and its potency in blocking bacterial growth isenhanced approximately five-fold by the presence of a GalNAc residue atThr11.³ A similar glycosylation-induced enhancement in potency isobserved for other drosocin glycoforms,^(3, 4) suggesting that theglycan exerts a conformational influence on the peptide.

Formulae 1a. Native drosocin (1), bearing a proximal GalNAc residueattached in an α-glycosidic linkage to Thr11, and a drosocinneoglycopeptide (2) bearing an oxime-linked α-GalNAc residue. Therecited amino acid sequence, “GKPRPYSPRP”, is SEQ ID NO:2, and thesequence “SHPRPIRV” is SEQ ID NO:3.

We envisioned that the oxime-linked neoglycopeptide (2) could beobtained from the highly selective reaction of an aminooxy sugar with apeptide bearing an unnatural ketone side chain. The ketone group ischemically orthogonal to all natural amino acid side chains and reactswith aminooxy groups in a highly specific manner, allowing site-specificconjugation without the requirement for protecting groups on the sugaror peptide. While methods for the synthesis of neoglycopeptides bearingother non-native linkages are well known,⁵ the majority of these methodsinvolve the coupling of electrophilic carbohydrate derivatives withnucleophilic amino acids. For example, bromoacetamides^(5d) orisothiocyanates^(5e) can be coupled with exposed cysteine or lysineresidues, respectively. However, in the presence of several copies ofthese nucleophilic residues, such methods are not site selective.

As described elsewhere herein, unprotected ketone groups can beincorporated into a peptide using Fmoc-protected (2S)-aminolevulinicacid (3) by solid-phase peptide synthesis (SPPS) (Scheme 1a). Wegenerated keto-drosocin (4) in this fashion, in which the(2S)-aminolevulinic acid residue replaced Thr11, the natural site ofglycosylation.

The aminooxy sugar, α-GalNAc derivative 7 (Scheme 2), was generated fromglycosyl chloride 5⁷ using a method similar to that reported by Roy andcoworkers.⁸ Compound 5 was reacted with N-hydroxysuccinimide (NHS) toafford the α-NHS glycoside 6. Reductive acetylation of the azide,followed by deprotection of the acetyl esters and succinimido groupprovided the desired aminooxy sugar (7).⁹

Reagents and conditions: (a) N-hydroxysuccinimide, Bu₄N(HSO₄), 1:1CH₂Cl₂/1 M Na₂CO₃, 52%; (b) H₂, 10% Pd/C, Ac₂O, 100%; (c) H₂N₄

DH₂O, 71%.

The coupling reaction of keto-drosocin (4) with aminooxy GalNAc (7) wascarried out in 1.0 M NaOAc buffer, pH 5.5 at 37° C. (Formulae 2a). Theprogress of the reaction was monitored by reversed-phase HPLC. Thereaction was essentially complete after eight hours, as indicated by thepresence of a single peak in the HPLC trace. No significant byproductswere observed, and the identity of the drosocin neoglycopeptide (2) wasconfirmed by electrospray ionization mass spectrometry (ESI-MS).

In order to evaluate the functional consequences of replacing theglycosidic linkage at Thr11 with an unnatural oxime linkage, weevaluated the bacteriostatic activity of neoglycopeptide 2 and comparedits activity to both native (1) and unglycosylated drosocin.¹⁰ Theoxime-linked neoglycopeptide (2) was found to be four-fold more potentin blocking bacterial growth (IC₅₀=0.16±0.04 μM) than unglycosylateddrosocin (IC₅₀=0.63±0.05 μM), and similar in potency to native drosocin(1) (IC₅₀=0.10±0.02 μM). These results indicate that the nativesugar-peptide linkage in drosocin is not essential to achieveglycosylation-dependent enhancement in potency. Analagous results areobtained with other O-linked glycopeptides.

Formulae 2a. Coupling reaction of keto-drosocin (4) with aminooxy GalNAc(7) to give oxime-linked neoglycopeptide 2. The recited amino acidsequence, “GKPRPYSPRP”, is SEQ ID NO:2, and the sequence “SHPRPIRV” isSEQ ID NO:3.

In summary, we have shown that a functional neoglycopeptide can besynthesized by the condensation of an aminooxy sugar with aketo-peptide. There are several convenient features of this approach.First, this method obviates the need for the cumbsersome assembly ofglycosylated amino acids typically used in the synthesis of nativeO-linked glycopeptides.¹¹ Second, the carbohydrate can be installed at auser-defined location within any given peptide without concern fordifferential protection of amino acid side chains. Third, the highlyselective coupling reaction is carried out under aqueous conditionswithout use of auxiliary coupling reagents. Furthermore, the onlybyproduct of the reaction is water, minimizing the need forpurification. Finally, a variety of neoglycopeptides can be obtainedfrom a single keto-peptide using this convergent synthetic strategy.

REFERENCES AND NOTES

1. (a) Anisfeld, S. T.; Lansbury, J. P. T. J. Org. Chem. 1990, 55,5560-5562. (b) Cohen-Anisfeld, S. T.; Lansbury, J. P. T. J. Am. Chem.Soc. 1993, 115, 10531-10537. (c) Roberge, J.; Beebe, X.; Danishefsky, S.J.; Science 1995, 269, 202-204. (d) Danishefsky, S. J.; Hu, S.; Cirillo,P. F.; Eckhardt, M.; Seeberger, P. Chem. Eur. J. 1997, 3, 1617-1628.

2. (a) Andreotti, A. H.; Kahne, D. J. Am. Chem. Soc. 1993, 115,3352-3353. (b) Liang, R.; Andreotti, A. H.; Kahne, D. J. Am. Chem. Soc.1995, 117, 10395-10396. (c) Maeji, N. J.; Inoue, Y.; Chûjô, R.Biopolymers 1987, 26, 1753-1767.

3. (a) Bulet, P.; Urge, L.; Ohresser, S.; Hetru, C.; Otvos, L. Eur. J.Biochem. 1996, 238, 64-69. (b) Bulet, P.; Dimarcq, J. -L.; Hetru, C.;Languex, M.; Charlet, M.; Hegy, G.; Dorsselar, A. V.; Hoffman, J. A. J.Biol. Chem. 1993, 268, 14893-14897.

4. Rodriguez, E. C.; Winans, K. A.; King, D. S.; Bertozzi, C. R. J. Am.Chem. Soc. 1997, 119, 9905-9906.

5. (a) Stowell, C. P.; Lee, Y. C. Neoglycoproteins. The Preparation andApplication of Synthetic Glycoproteins; Academic Press: San Francisco,1980; Vol. 37, pp 225-281. (b) Mangusson, G.; Chernack, A. Y.; Kihlberg,J.; Kononov, L. O. Synthesis of Neoglycoconjugates; Academic Press: SanDiego, 1994. (c) Lee, Y. C.; Lee, R. T. Meth. Enzymol. 1994, 242, 3-123.(d) Wong, S. Y. C.; Guile, G.; Dwek, R.; Arsequell, G. Biochem. J. 1994,300, 843-850. (e) Mulins, R. E.; Langdon, R. G. Biochemistry 1980, 19,1199-1205.

6. Marcaurelle, L. A.; Bertozzi, C. R., preceeding paper.

7. Lemieux, R. U.; Ratcliffe, R. M. Can. J. Chem. 1979, 57, 1244-1251.

8. Cao, S.; Tropper, F. D.; Roy, R. Tetrahedron 1995, 51, 6679-6686.

9. Characterization ofaminooxy-2-acetamido-2-deoxy-α-D-galactopyranoside (7): ¹H NMR (300 MHz,D₂O): δ 4.97 (d, 1 H, J=4.0), 4.23 (dd, 1 H, J=11.3, 4.0), 4.00 (m, 2H), 3.81 (m, 3 H), 2.06 (s, 3 H); ¹³C NMR (100 MHz,): δ 174.55, 100.55,70.93, 68.34, 67.49, 61.07, 49.11, 21.83; FAB-HRMS calcd for C₈H₁₇N₂O₆(M+H⁺) 237.1087, found 237.1084.

10. The growth inhibition assay was performed essentially as describedby Bulet et al.³ Sterile 96-well plates were used, with a final volumeof 100 μL per well. This volume consisted of 90 μL of mid-logarithmicphase culture of E. coli D22 in LB media containing streptomycin (50μg/mL), added to 10 μL of serially diluted peptide (native drosocin (1),unglycosylated drosocin or neoglycopeptide 2) in water. Final peptideconcentrations ranged from 10⁻⁸ to 10⁻⁵ M. Plates were incubated for 24h at rt. Bacterial growth was determined by measuring the absorbance at415 nm on a BioRad 550 microplate reader. Measurements were taken at t=0h and t=24 h, and the change in absorbance (optical density) wasrecorded.

11. (a) Meldal, M.; St. Hiliare, P. M. Curr. Opin. Chem. Biol. 1997, 1,552-563. (b) Mathieux, N.; Paulsen, H.; Meldal, M.; Block, K. J. Chem.Soc., Perkin Trans. 1 1997, 2259-2364. (c) Sames, D.; Chen, X. -T.;Danishefsky, S. J. Nature 1997, 389, 587-591.

EXAMPLE III Aminooxy, hydrazide and theosemicarbazide-functionalizedsaccharides: versatile reagents for glycoconjugate synthesis.

ABSTRACT: Saccharides functionalized at their reducing termini withaminooxy, hydrazide or thiosemicarbazide groups are versatile reagentsfor the synthesis of glycoconjugates using chemoselective ligationreactions. They can be prepared from aminoglycosides without therequirement for protecting groups on the saccharide. Novelneoglycopeptides were constructed by the condensation of aminooxy andthiosemicarbazide-functionalized oligosaccharides with ketone groups onan unprotected peptide scaffold. In this fashion, we synthesizedneoglycopeptides bearing the chitobiosyl moiety found in N-linkedglycopeptides, or a sialyl Lewis x motif found in O-linkedglycopeptides.

Organic chemists have exercised significant creativity in theconstruction of glycoconjugate assemblies as tools for studyingcarbohydrate recognition and as potential therapeutic agents.¹ Thesesynthetic accomplishments, which include neoglycoproteins²,glycodendrimers³, glycoliposomes⁴ and glycopolymers⁵, have been sparkedby the growing realization that glycoconjugates participate in a widerange of normal and pathophysiological processes.⁶ Given the importanceof glycoconjugates as tools for glycobiology and as emergingpharmaceutical reagents, new methods for attaching sugars to scaffoldsare of significant current interest.

The highly selective condensation reactions of ketones or aldehydes withaminooxy, hydrazide or thiosemicarbazide groups (forming oximes,hydrazones and thiosemicarbazones, respectively) are popular for theconjugation of peptides and proteins.^(7,8) The reactions proceed inaqueous solvent and their high selectivity obviates the requirement forprotection of other functional groups on the coupling partners. Despitethe utility of these ‘chemoselective ligation’ reactions^(7,8) in theassembly of peptide-based macromolecules, their implementation inglycoconjugate synthesis is limited to a few examples.⁹ The majority ofcurrent methods for attaching sugars to scaffolds involve the couplingof electrophilic carbohydrate derivatives with exposed thiol or aminogroups. The electrophilic derivatives include α-haloacetamides,¹⁰bromoethyl glycosides,¹¹ maleimides,¹² and isothiocyanates.¹³ Here wereport an alternate ligation strategy based on the coupling ofnucleophilic carbohydrate derivatives to synthetic scaffolds. Wesynthesized carbohydrates bearing aminooxy, hydrazide orthiosemicarbazide groups at their reducing termini, and coupled two ofthese derivatives to ketone groups on a peptide scaffold. The novelneoglycopeptides produced in this fashion have structural motifs sharedby native N-linked or O-linked glycopeptides.¹⁴

The direct attachment of an aminooxy group to the reducing terminus of amono- or disaccharide can be accomplished by formation of theN-hydroxysuccinimido (NHS) glycoside¹⁵ followed by cleavage of thesuccinimide group with hydrazine, a strategy first reported by Roy andcoworkers.^(15b) Accordingly, we synthesized β-linked aminooxy analogsof galactose (4), N-acetylglucosamine (GlcNAc) (5) and lactose (6) fromthe corresponding protected NHS glycosides 1, 2 and 3, respectively(Scheme 1b). In addition, we prepared an α-aminooxy GalNAc derivative(8) to mimic of the peptide-proximal α-GalNAc residue found in O-linkedglycoproteins. This was achieved by first generating the α-NHS glycosideof peracetylated 2-azido-2-deoxy galactose by reaction of azido chloride7¹⁶ with NHS. Subsequent reductive acetylation of the azide anddeprotection with hydrazine furnished the desired analog.¹⁷

The simple aminooxy sugars 4-6 and 8 can be transformed into morecomplex oligosaccharides using established enzymatic methods.¹⁸ As anexample, we synthesized an aminooxy-functionalized analog of thetetrasaccharide sialyl Lewis x (NeuAcα2ø3Galβ1ø4(Fucα1ø3)GlcNAc), amotif recognized by the selectin family of adhesion molecules that hasbeen explored as a selectin inhibitor in the form of many differentconjugated assemblies.¹⁹ Aminooxy lactose (6) was converted to thecorresponding sialyllactose analog using an α(2,3)-sialyltransferase(α(2,3)-ST) and the glycosyl donor CMP-sialic acid. Without isolation,aminooxy sialyllactose was fucosylated using anα(1,3)-fucosyltransferase (α(1,3)-FucT) with GDP-fucose as the glycosyldonor affording sialyl Lewis x analog 9 (Scheme 2b).^(20,21) Thepresence of the aminooxy group did not adversely affect the course ofthe enzymatic reactions.

In order to generate complex oligosaccharide coupling partners withoutthe use of glycosyltransferases, we required a strategy forfunctionalizing the reducing terminus with minimal protecting groupmanipulations. We therefore developed methods for the synthesis ofaminooxy, hydrazide and thiosemicarbazide-functionalized sugars thatemploy the well-known Kochetkov procedure²² for generatingglycosylamines from unprotected free-reducing sugars. β-Amino lactose(10) was prepared by stirring the free disaccharide in a concentratedsolution of NH₄HCO₃.^(22b) Aminooxy and hydrazide groups were thenintroduced as depicted in Scheme 3b. Compound 10 was coupled withN-(t-butoxycarbonyl)aminooxyacetic acid and subsequently deprotectedwith TFA to give aminooxy sugar 12. The coupling reaction of 10 withmonomethyl succinate, followed by treatment with hydrazine gavehydrazide 14.

The most efficiently prepared saccharide derivatives for chemoselectiveligation reactions were glycosyl thiosemicarbazides, prepared from thecorresponding isothiocyanates²³ as shown in Scheme3b. β-Amino lactose(10) was reacted with thiophosgene²⁴ in aqueous solution to give thecorresponding isothiocyanate (16). Without isolation, the isothiocyanate(16) was immediately converted to thiosemicarbazide 18 by treatment withhydrazine. The same procedure when applied to β-amino chitobiose(15)^(24a) furnished thiosemicarbazide 19. The overall yields of theisolated thiosemicarbazides ranged from 50-70%. The synthesis ofcompound 19 highlights the utility of this procedure for transformingsmall amounts of precious oligosaccharides into suitable couplingpartners.

Finally, we constructed neoglycopeptides containing motifs found innaturally occurring N- and O-linked glycopeptides by coupling thenucleophilic sugars to a synthetic peptide fashioned with a ketone group(20, Formulae 1b). Keto-peptide 20 was synthesized by the directincorporation of (2S)-aminolevulinic acid into the peptide duringFmoc-based solid-phase synthesis, a procedure we have recentlyreported.²⁴ We reacted keto-peptide 20 with chitobiose thiosemicarbazide(19) to afford neoglycopeptide 21, a structure that resembles the corechitobiosyl-asparagine motif shared by all N-linked glycoproteins(Formulae 1b).²⁵ The coupling reaction of keto-peptide 20 with aminooxysialyl Lewis x analog 9 produced neoglycopeptide 22, which resembles theO-linked glycopeptides that function as native selectin ligands.²⁶ Itshould be emphasized that these condensation reactions proceed inaqueous solvent without need for auxiliary reagents. In addition,subsequent purification is straightforward as the only other product ofthe reaction is water. The facile construction of neoglycopeptidesrelated to native N- and O-linked structures underscores the utility ofthis approach for glycoconjugate synthesis.

Synthetic procedures for compounds 4-6, 8, 9, 12, 14, 18 and 19:

General methods: All chemical reagents were obtained from commercialsuppliers and used without further purification.α(2,3)-Sialyltransferase, α(1,3)-fucosyltransferase, CMP-sialic acid andGDP-fucose were purchased from Calbiochem. Flash chromatography wasperformed using 230-400 mesh silica gel 60 (E. Merck No 9385).Analytical thin layer chromatography (tlc) was conducted on AnaltechUniplate silica gel plates with detection by ceric ammonium molybdate,p-anisaldehyde, ninhydrin and/or UV light. Unless otherwise specified,all reactions were conducted at rt. High-pressure liquid chromatography(HPLC) was performed on a Rainin Dynamax SD-200 system using Microsorband Dynamax aminopropyl and C₁₈ columns (particle size 5 μm; analyticalcolumn: 4.6 mm ID×25 cm, 1 mL/min; semi-preparative column: 10 mm ID×25cm, 3 mL/min), and UV detection (200 or 230 nm) was performed with aRainin Dynamax WV-1 detector. It is important to note that use of lowwavelengths (200 nm) was important for detecting aminooxy sugarderivatives which have very low extinction coefficients. The ¹H- and¹³C-NMR spectra were obtained at either 400 or 500 MHz with BrukerAMX-400 and DRX-500 spectrometers. Chemical shifts are reported in δvalues downfield from tetramethylsilane (TMS) and coupling constants arereported in Hz.

Aminooxy-β-D-galactopyranoside (4). Compound 1 (1.0 g, 2.2 mmol) wasdissolved in 25 mL of N₂H₄.H₂O_(x) (55%) and the solution was stirred atrt overnight. The reaction mixture was concentrated in vacuo, filteredand purified by HPLC on aminopropyl silica gel (200 nm detection) usinga gradient of CH₃CN:H₂O (100:0ø55:45 over 60 min) to provide 0.38 g(86%) of 4. (The product has low UV absorbtivity relative to the otherproducts, butanedioic hydrazide and acetyl hydrazide, and tends to eluteclosely following butanedioic hydrazide during HPLC purification).¹H-NMR (400 MHz, D₂O): δ 3.50 (app t, 1 H, J=9.0), 3.65 (dd, 1 H, J=6.8,9.9), 3.70 (m, 3 H), 3.90 (d, 1 H, J=3.3), 4.50 (d, 1 H, J=8.1); ¹³C-NMR(100 MHz, D₂O): δ 63.58, 71.14, 71.89, 75.35, 77.65, 108.11; FAB-HRMS(M+H⁺) calcd for C₆H₁₄NO₆ 196.0821, found 196.0819.

Aminooxy-2-acetamido-2-deoxy-β-D-glucopyranoside (5). Compound 2 (100mg, 0.2 mmol) was dissolved in 20 mL of N₂H₄.H₂O (55%) and the solutionwas stirred at rt overnight. The reaction mixture was concentrated invacuo, filtered and purified by aminopropyl silica gel HPLC (200 nmdetection) using a gradient of CH₃CN:H₂O (100:0ø60:40 over 75 min) toprovide 29 mg (55%) of 5. ¹H-NMR (500 MHz, D₂O): δ 2.01 (s, 3 H), 3.43(m, 2 H), 3.52 (app t, 1 H, J=8.6), 3.69 (dd, 1 H, J=9.1, 10.5), 3.73(dd, 1 H, J=5.5, 12.3), 3.91 (dd, 1 H, J=1.8, 12.2), 4.75 (d, 1 H,J=8.8); ¹³C-NMR (125 MHz, D₂O): δ 22.01, 53.74, 60.58, 69.67, 73.69,75.71, 103.55, 174.69; FAB-HRMS (M+H⁺) calcd for C₈H₁₇N₂O₆ 237.1087,found 237.1082.

Aminooxy-β-D-lactoside (6). Compound 3 (0.50 g, 0.68 mmol) was dissolvedin 6.8 mL of N₂H₄.H₂O_(x) (55%) and the solution was stirred at rtovernight. The reaction was concentrated in vacuo, filtered and purifiedby aminopropyl silica gel HPLC (200 nm detection) using a gradient ofCH₃CN:H₂O (100:0ø50:50 over 90 min) to provide 0.20 g (81%) of 6. ¹H-NMR(500 MHz, D₂O): δ 3.33 (app t, 1 H, J=9.2), 3.52 (dd, 1 H, J=7.8, 9.9),3.81-3.60 (m, 8 H), 3.90 (d, 1 H, J=3.3), 3.98 (dd, 1 H, J=2.1, 12.3),4.42 (d, 1 H, J=7.8), 4.57 (d, 1 H, J=8.3); ¹³C-NMR (100 MHz, D₂O): δ60.15, 61.12, 68.65, 71.05, 71.46, 72.62, 74.53, 74.81, 75.45, 78.30,103.03, 104.94; FAB-HRMS (M+H⁺) calcd for C₁₂H₂₄NO₁₁ 358.1349, found358.1350.

2-Azido-2-deoxy-3,4,6-tri-O-acetyl-α-D-galactopyranosylhydroxysuccin-imide (8a). To a solution of N-hydroxysuccinimide (1.3 g,7.5 mmol) and tetrabutylammonium hydrogensulfate (0.79 g, 2.3 mmol) in 1M Na₂CO₃ (11.5 mL) was added a solution of 7 (0.81 g, 2.3 mmol) inCH₂Cl₂ (11.5 mL). The biphasic mixture was stirred vigorously for 24 h,diluted with CH₂Cl₂ (50 mL), and washed with H₂O (2×25 mL) and brine (40mL). The aqueous layers were extracted with CH₂Cl₂, and the combinedorganic layers were dried (Na₂SO₄) and concentrated in vacuo. The crudeproduct was purified by silica gel chromatography (100:1 CHCl₃/MeOH) toyield 0.41 g (52%) of 8a as mixture of anomers (4:1 α/β). The α-anomerwas isolated as a white solid upon trituration with CH₂Cl₂/hexanes.¹H-NMR (500 MHz, CDCl₃): δ 2.05 (s, 3 H), 2.07 (s, 3 H), 2.15 (s, 3 H),2.77 (s, 4 H), 3.92 (m, 2 H), 4.21 (dd, 1 H, J=6.5, 11.3), 5.09 (app t,1 H, J=7.2), 5.45 (dd, 1 H, J=3.2, 11.5), 5.52 (d, 1 H, J=3.7), 5.56(dd, 1 H, J=1.4, 3.1); ¹³C-NMR (125 MHz, CDCl₃): δ 22.11, 22.22, 26.81,57.10, 61.59, 67.56, 67.91, 69.18, 101.51, 167.00, 167.32, 167.80,167.86; FAB-MS: Calcd. for Cl₆H₂₀N₄O₁₀ 428, found m/z 429 (M+H⁺). Anal.Calcd. for C₁₆H₂₀N₄O₁₀: C, 44.86; H, 4.71; N, 13.08. Found: C, 44.82; H,4.54; N, 13.03.

2-Acetamido-2-deoxy-3,4,6-tri-O-acetyl-α-D-galactopyranosylhydroxy-succinimide (8b). To a solution of 8a (56 mg, 0.13 mmol) and 10%Pd/C (11 mg) in 1:1 MeOH/CH₂Cl₂ (3 mL) was added acetic anhydride (25μL, 0.26 mmol). The mixture was stirred under an atmosphere of H₂ gas atrt. After 3.5 h, the reaction mixture was filtered through Celite andconcentrated in vacuo to give 58 mg (100%) of compound 8b. ¹H-NMR (500MHz, CDCl₃): δ 2.00 (s, 3 H), 2.04 (s, 3 H), 2.07 (s, 3 H), 2.15 (s, 3H), 2.73 (s, 4 H), 3.93 (dd, 1 H, J=6.7, 11.4), 4.25 (dd, 1 H, J=5.9,11.4), 4.73 (td, 1 H, J=3.7, 9.6),4.97 (t, 1 H, J=6.0), 5.28 (m, 2 H),5.51 (d, 1 H, J=1.9), 6.10 (d, 1 H, J=9.5); ¹³C-NMR (125 MHz, CDCl₃): δ20.66, 20.72, 23.14, 25.37, 47.15, 61.59, 67.12, 67.31, 69.21, 104.20,170.11, 170.44, 170.58, 170.61, 170.86; FAB-HRMS (M+H⁺) calcd. forC₁₈H₂₄N₂O₁₁ 445.1458, found 445.1467.

Aminooxy 2-acetamido-2-deoxy-α-D-galactopyranoside (8). Compound 8b (57mg, 0.13 mmol) was dissolved in 5 mL of N₂H₄.H₂O_(x) (55%) and thesolution was stirred at rt overnight. The solution was concentrated invacuo, filtered and purified by aminopropyl silica gel HPLC (200 nmdetection) using a gradient of CH₃CN:H₂O (90:10ø65:35 over 60 min) toprovide 26 mg (85%) of 8. ¹H-NMR (500 MHz, CDCl₃): δ 2.06 (s, 3 H), 3.81(m, 3 H), 4.00 (m, 2 H), 4.23 (dd, 1 H, J=4.0, 11.3), 4.97 (d, 1 H,J=4.0); ¹³C-NMR(125 MHz, CDCl₃): δ 21.83, 49.11, 61.07, 67.49, 68.35,70.93, 100.54, 174.56; FAB-HRMS (M+H⁺) calcd for C₈H₁₇N₂O₆ 237.1087,found 237.1084.

Aminooxy sialyl Lewis x analog (9). To 5 mg of 7 (0.014 mmol) was addedin succession water (105 μL), 0.5 M HEPES (14 μL, pH 7.4), 25% TritonCF-54 (2.8 μL), and 5% BSA (2.2 μL). To this solution was addedCMP-sialic acid (10 mg, 0.014 mmol) followed by α(2,3)-sialyltransferase(12.5 μL, 1 mU/μL), and calf intestinal alkaline phosphatase (CIAP) (2.9μL, 1 U/μL, Sigma). The reaction was incubated for 5 d at rt. To thereaction mixture was then added water (18 μL), 0.5 M HEPES (5 μL, pH7.4), 1.0 M MnCl₂ (1 μL), and GDP-fucose (9 mg, 0.014 mmol).α(2-3)-Fucosyltransferase (E.C. 2.4.1.65) (25 mL, 0.5 mU/mL) and ClAP (1μL) were added and the reaction was incubated at rt for an additional 5d. The solution was filtered through an Amicon 3 kDa membrane andpurified by aminopropyl silica gel HPLC (195 nmn detection) eluting with74:26 CH₃CN/15 mM KH₂PO₄, pH 5.2, to provide compound 9. Due to thepresence of buffer salts in the eluant, an isolated yield could not beaccurately determined, but HPLC analysis of the enzymatic reactionsindicated approximately 60% conversion to 9. ¹H-NMR (500 MHz, D₂O): δ1.17 (d, 1H, J=6.7), 1.79 (t, 1 H, J=12.1), 2.03 (s, 3 H, NHCOCH₃), 2.76(dd, 1 H, H-3″_(eq), J_(3″eq,4″), 4.6, J_(3″eq,3″ax)=12.5), 3.34 (br t,1 H, H-2), 3.94 (d, 1 H, H-4′, J_(4′,3′)=3.3) 4.02 (dd, 1 H, H-6_(a),J_(6a,5)=2.1, J_(6a,6b)=12.3), 4.08 (dd, 1 H, H-3′, J=3.2,J_(3′,2′)=10.0), 4.49 (d, 1 H, H-1, J_(1,2)=7.8), 4.59 (d, 1 H, H-1′,J_(1′,2′)=8.3), 5.43 (d, 1 H, J=4.0).

Compound 11. To a solution of N-(t-butoxycarbonyl)aminooxyacetic acid(31 mg, 0.16 mmol) in DMSO (1 mL) was added DIEA (76 mL, 0.44 mmol),compound 10 (50 mg, 0.15 mmol), and BOP (80 mg, 0.17 mmol) insuccession. The clear yellow solution was stirred at rt for 2 h. Thecrude product was precipitated from solution by the addition of 1:2acetone/ether (12 mL), incubation at −20° C. for 15 min andcentrifugation. A second batch of product was obtained by precipitationfrom the concentrated supernatant. The pooled precipitate was purifiedby aminopropyl silica gel HPLC (200 nm detection) using a gradient ofCH₃CN:H₂O (100:0ø35:65 over 60 min) to provide 31 mg (41%) of compound11. ¹H-NMR (500 MHz, D₂O): δ 1.48 (s, 9 H), 3.54 (m, 2 H), 3.75 (m, 9H), 3.94 (m, 2 H), 4.47 (m, 3 H), 5.07 (d, 1 H, J=9.2); ¹³C-NMR (125MHz, D₂O): δ 27.30, 58.78, 60.98, 68.42, 70.86, 71.43, 72.42, 74.54,74.90, 75.29, 76.43, 77.66, 78.77, 84.02, 84.02, 102.79, 158.60, 172.34;FAB-HRMS (M+H⁺) calcd for C₁₉H₃₅N₂O₁₄ 515.2088, found 515.2090.

Compound 12. Compound 11 (27 mg, 0.053 mmol) was dissolved in 3:2CH₂Cl₂/TFA (2 mL) and stirred at rt for 1.5 h. The solvent was removedunder vacuum and crude product was dissolved in water, neutralized usingweak anion exchange resin (Amberlyst) and concentrated to afford 21 mg(100%) of 12. ¹H-NMR (500 MHz, D₂O): δ 3.56 (m, 2 H), 3.77 (m, 11 H),3.94 (m, 2 H), 4.29 (d, 2 H, J=0.8), 4.47 (d, 1 H. J=2.6) 5.08 (d, 1 H,J=9.2); ¹³C-NMR (125 MHz, D₂O): δ 59.71, 60.96, 68.46, 70.84, 71.29,72.39, 73.47, 74.90, 75.26, 76.40, 77.58, 78.79, 102.77, 173.82;FAB-HRMS (M+H⁺) calcd for C₁₄H₂₇N₂O₁₂ 415.1564, found 415.1559.

Compound 13. To a solution of monomethylsuccinate (22 mg, 0.16 mmol) inDMSO (1 mL) was added DIEA (76 mL, 0.44 mmol), glycosylamine 10 (50 mg,0.15 mmol), and BOP (80 mg, 0.17 mmol) in succession. The clear yellowsolution was stirred at rt for 2 h. The crude product was precipitatedfrom solution by the addition of 1:2 acetone/ether (12 mL), incubationat −20° C. for 15 min and centrifugation. The pooled precipitate waspurified by aminopropyl silica gel HPLC (200 nm detection) using agradient of CH₃CN:H₂O (100:0ø35:65 over 60 min) to provide 32 mg (48%)of compound 13. ¹H-NMR (500 MHz, D₂O): δ 2.68 (m, 4 H), 3.45 (app t, 1H, J=9.1), 3.56 (dd, 1 H, J=7.8, 9.8), 3.74 (m, 14 H), 3.94 (m, 2 H),4.46 (d, 1 H, J=7.8) 4.99 (d, 1 H, J=9.2); ¹³C-NMR (125 MHz, D₂O): δ28.71, 30.08, 52.27, 59.81, 60.98, 68.49, 70.88, 71.41, 72.44, 75.00,75.29, 76.28, 77.72, 79.04, 102.80, 175.58, 176.01; HRMS (M+H⁺) calcdfor C₁₅H₂₈N₄O₁₂ 456.1704, found 456.1708.

Lactose succinic hydrazide (14). Compound 13 (31 mg, 0.67 mmol) wasdissolved in 2 mL of N₂H₄.H₂O_(x) (55%) and the solution was stirred atrt overnight. The reaction was concentrated in vacuo, filtered andpurified by aminopropyl silica gel HPLC (200 nm detection) using agradient of CH₃CN:H₂O (90: 10ø65:35 over 60 min) to provide 18 mg (59%)of 14. ¹H-NMR (500 MHz, D₂O): δ 2.52 (app t, 2 H, J=6.9), 2.64 (m, 2 H),3.44 (app t, 1 H, J=9.0), 3.56 (dd, 1 H, J=7.9, 8.8), 3.75 (m, 14 H),3.94 (m, 3 H), 4.46 (d, 1 H, J=7.7) 4.98 (d, 1 H, J=9.2); ¹³C-NMR (125MHz, D₂O): δ 28.54, 30.63, 59.78, 60.97, 68.47, 70.86, 71.39, 72.42,74.96, 75.28, 76.28, 77.67, 79.01, 102.78, 173.62, 175.85; FAB-HRMS(M+H⁺) calcd for C₁₆H₃₀N₃O₁₂ 456.1829, found 456.1826.

Lactose thiosemicarbazide (18). To a solution of 10 (50 mg, 0.15 mmol)in 3 mL of 0.3 M NaHCO₃ was added thiophosgene (33 μL, 0.44 mmol). Theorange solution was stirred at rt for 20 min during which the glycosylisothiocyanate (16) was produced in quantitative yield as determined bytlc analysis. To this solution was added 26 μL (0.45 mmol) ofN₂H₄.H₂O_(x) (55%) resulting in a bright yellow solution. This solutionwas stirred for 20 min at rt then concentrated in vacuo. Subsequent HPLCpurification on aminopropyl silica gel [CH₃CN:H₂O (90:10ø65:35 over 60min)] afforded 29 mg (48%) of thiosemicarbazide 18. ¹H-NMR (500 MHz,D₂O): δ 3.57 (dd, 1 H, J=7.8, 9.9), 3.76 (m, 9 H), 3.95 (m, 2 H), 4.48(d, 1 H, J=7.8) 5.54 (br d, 1 H, J=8.5); ¹³C-NMR (125 MHz, D₂O): δ59.71, 60.98, 68.49, 70.87, 71.58, 72.42, 74.92, 75.28, 76.20, 77.79,83.24, 102.82; HRMS (M+H⁺) calcd for C₁₃H₂₆N₃O₁₀S 416.1339, found416.1338. Appropriate precautions should be taken to avoid contact withthiophosgene; it is a highly toxic and corrosive reagent.

Chitobiose thiosemicarbazide (19). To a solution of β-amino chitobiose15 (9.0 mg, 0.021 mmol) in 0.3 M NaHCO₃ (0.5 mL) was added thiophosgene(5.1 μL, 0.067 mmol). The orange solution was stirred at rt for 30 minto give the glycosyl isothiocyanate (17) in quantitative yield asdetermined by tlc analysis. To the solution was then added 3.9 μL (0.070mmol) of N₂H₄.H₂O_(x) (55% ) resulting in a bright yellow solution. Thissolution was stirred for 10 min at rt then concentrated in vacuo.Subsequent HPLC purification on aminopropyl silica gel [CH₃CN:H₂O (90:10ø65:35 over 60 min)] afforded 6.6 mg (62%) of thiosemicarbazide 19.¹H-NMR (500 MHz, D₂O): δ 2.00 (s, 3 H), 2.07 (s, 3 H), 3.72 (m, 12 H),4.61 (d, 1 H, J=8.5) 5.52 (d, 1 H, J=9.6); ¹³C-NMR (125 MHz, D₂O): δ21.79, 22.01, 53.76, 55.50, 59.91, 60.44, 69.62, 72.34, 73.34, 75.80,75.93, 78.95, 82.52, 101.36, 174.55, 174.86; FAB-HRMS (M+H⁺) calcd forC₁₇H₃₂N₅O₁₀S 498.1870, found 498.1869. Appropriate precautions should betaken to avoid contact with thiophosgene; it is a highly toxic andcorrosive reagent

Synthesis of neoglycopeptides 21 and 22. To 50 μL of keto-peptide 20 (50mM) was added 10 μL of 1 M NaOAc buffer (pH 5.5) and 40 μL of 19 or 9(100 mM). The reaction mixture was incubated at 37 C for 24 h and thecorresponding product (21 or 22, respectively) was isolated by RP-HPLCwith a gradient of CH₃CN: H₂O, both with 0.1% TFA (10:90 0 30:60 over 30min). ESI-MS: Glycopeptide 21: calcd 2690.0 (M+H⁺), found 2986.9.Glycopeptide 22: calcd 2987.3 (M+H⁺), found 2986.9, calcd 3009.3(M+Na⁺), found 3009.1.

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12. Hansen, P. R.; Olsen, C. E.; Holm, A. Biooconjugate Chem. 1998, 9,126.

13. (a) Mullins, R. E.; Langdon, R. G. Biochemistry 1980, 19, 1199; (b)For a review on glycosyl isothiocyanates see, Witczak, Z. J. Adv.Carbohydr. Chem. Biochem. 1986, 44, 91.

14. For a review on the synthesis of native glycopeptides see, Large, D.G.; Warren, C. D. Glycopeptides and Related Compounds: Synthesis,Analysis, and Applications; Marcel Dekker: New York, 1997.

15. (a) Andersson, M.; Oscarson, S. Glycocnjugate J. 1992, 9, 122; (b)Cao, S.; Tropper, F. D.; Roy, R. Tetrahedron 1995, 51, 6679.

16. Lemieux, R. U.; Ratcliffe, R. M. Can. J. Chem. 1979, 57,1244.

17. Saccharides with hydrazine attached at the reducing terminus, orglycosylhydrazines, have also been reported. They react with aldehydesto form hydrazones, but the glycosidic linkage hydrolyzes in water afterseveral hours. By contrast, we have found aminooxy sugars to be stablefor months in aqueous solution. (a) Takasaki, S.; Mizuochi, T.; Kobata,A. Meth. Enzymol. 1982, 83, 263; (b) Tolvanen, M.; Gahmberg, C. G. J.Biol. Chem. 1986, 261, 9546.

18. (a) Palcic, M. M. Meth. Enzymol. 1994, 230, 300; (b) McGarvey, G.J.; Glenn, J.; Wong, C. -H. Liebigs Ann. 1997, 1059.

19. Simanek, E. E.; McGarvey, G. J.; Jablonowski, J. A.; Wong, C. -H.Chem. Rev. 1998, 98, 833.

20. The enzymatic reactions were executed essentially as described inIchikawa, Y.; Lin, Y. -C.; Dumas, D. P.; Shen, G.-J.; Garcia-Junceda,E.; Williams, M. A.; Bayer, R.; Ketcham, C.; Walker, L. E.; Paulson, J.C.; Wong, C. -H. J. Am. Chem. Soc. 1992, 114, 9283.

21. The α(2,3)-ST and α(1,3)-FucT were purchased from Calbiochem.

22. (a) Likhosherstov, L. M.; Novikova, O. S.; Derevitskaja, V. A.;Kochetkov, N. K. Carbohydr. Res. 1986, 146, CI; (b) Vetter, D.; Gallop,M. A. Bioconjugate Chem. 1995, 6, 316; (c) Meinjohanns, E.; Meldal, M.;Paulsen, H.; Dwek, R. A. J. Chem. Soc., Perkin Trans. 1 1998, 549.

23. Unprotected glycosyl isothiocyanates have been used for proteinmodification (ref. 13), but their conversion to glycosylthiosemicarbazides has not to our knowledge been reported.

24. Marcaurelle, L. A.; Bertozzi, C. R., Tetrahedron Lett., in press.

25. Coupling reactions were performed in 1 M NaOAc buffer, pH 5.5, 37°C. The neoglycopeptide products were purified by RP-HPLC andcharacterized by ES-MS. The yields of the coupling reactions ranged from85 to 95%.

26. Rosen, S. D.; Bertozzi, C. R. Curr. Biol. 1996, 6, 261.

EXAMPLE IV A strategy for the chemoselective synthesis of )-linkedglycopeptides with native sugar-peptide linkages.

ABSTRACT: The convergent coupling of oligosaccharides to a proteinscaffold is an attractive approach to the synthesis of complexglycopeptides. Here we describe a strategy for the convergent assemblyof O-linked glycopeptide analogs using the principle of chemoselectiveligation. A single GalNAc residue was incorporated into a glycopeptideby solid phase methods, and then oxidized to the corresponding aldehydewith the enzyme galactose oxidase. Hydroxylamine-functionalized sugarswere ligated onto the glycopeptide aldehyde affording oxime-linkedproducts with native sugar-peptide linkages. This strategy wasdemonstrated in the synthesis of chemoselectively ligated analogs of theantibacterial glycopeptide drosocin.

The critical role of specific oligosaccharide structures in thebiological function of many glycoproteins is now well appreciated.¹ Theimportance of protein-bound oligosaccharides in cell-cell recognitionevents,² and in modulating protein folding and stability³ has beenhighlighted in a number of recent landmark studies, inspiring thedevelopment of new synthetic methods for the construction ofglycoproteins with defined, homogeneous glycoforms. Many of thedifficulties inherent to the synthesis of such complex molecules,including the requirement for extensive protecting group manipulationsand the chemical sensitivity of glycosidic linkages, have been elegantlyaddressed by several groups.^(4,5) Yet, the convergent coupling oftailor-made oligosaccharides to a protein scaffold, an appealingstrategy for the synthesis of complex glycoproteins, has been successfulonly in the construction of the amide sugar-peptide linkage found inN-linked glycopeptides. The extension of this approach to O-linkedglycopeptides has been hindered by the difficulties endemic to theformation of a sugar-peptide glycosidic bond.

Here we report a strategy for the convergent synthesis of O-linkedglycopeptide analogs with native sugar-peptide linkages using theprinciple of chemoselective ligation.⁶ At the heart of this approach isthe introduction of mutually and uniquely reactive functional groups(e.g., an aldehyde group and a hydroxylamino group) onto unprotectedfragments and the coupling of these fragments in an aqueous environment.First, building block 1 [N^(α)-Fmoc-Thr/Ser(AC₃-α-D-GalNAc)] isincorporated into a glycopeptide by solid phase peptide synthesis(SPPS).^(4c-j) Next, a chemically unique functional group forchemoselective ligation is introduced using the commercially availableenzyme galactose oxidase,⁷ which selectively converts galactose orGalNAc residus to the corresponding C-6 aldehydes. The aldehyde groupsare reacted with an unprotected oligosaccharide bearing a hydroxylaminogroup at the reducing end, affording an oxime-linked analog of the β1ø6glycosidic linkage that is frequently observed in naturally occurringO-linked glycans.⁸ This approach allows flexibility in the elaborationof outlying glycoforms while retaining the native proximalGalNAc-α-Ser/Thr linkage.

Our focus on preserving the sugar-peptide linkage was motivated byseveral studies suggesting a major role for the proximal GalNAc residuein modulating local peptide conformation.⁹ In some glycopeptide targets,perturbation of this linkage might result in loss of native conformationand therefore function. Several methods are available for the covalentattachment of oligosaccharides to peptides through non-nativelinkages,¹⁰ including chemoselective ligation of the reducing terminalaldehyde of an oligosaccharide to an N-terminal hydroxylamino group.¹¹These methods may not be suitable, however, for the synthesis ofglycoproteins with glycosylation-dependent active conformations.

To demonstrate this methodology we selected the insect-derived,antibacterial 19-amino acid glycopeptide drosocin, the biologicalactivity of which is influenced by glycosylation.¹² Drosocin's potencyin blocking bacterial growth is enhanced 2-8 fold (depending on thetarget bacterial strain) by a single O-linked disaccharide (GaloGalNAc)at Thr11.¹³ Threonine derivative 1^(4h) was incorporated into drosocinusing Fmoc-based solid-phase methods to give GalNAc-drosocin 2, whichwas oxidized with galactose oxidase (Sigma) to the corresponding C-6aldehyde (isolated yields were >70%). Hydroxylamino derivatives ofgalactose (3) and GlcNAc (4) were prepared from the correspondingN-hydroxysuccinimidoglycosides using the method of Roy and coworkers.¹⁴Compounds 3 and 4 were coupled with the glycopeptide aldehyde to givechemoselectively ligated products 5 and 6, respectively (isolated yieldswere >80%). The glycosylation sequence of glycopeptide 5 mimics that ofnative drosocin. The glycan in glycopeptide 6 mimics the naturallyoccurring GlcNAcβ1ø6GalNAc (“core 6”) structure; an oxime groupsubstitutes for the natural glycosidic bond and the linkage is extendedby one atom.

Unprotected oligosaccharides, either synthetic or derived from naturalsources, can be converted to the corresponding glycosylamines viaKochetkov amination.^(4k,4l,15) Functionalization of these derivativeswith a hydroxylamino group would provide access to a wide variety ofoligosaccharide coupling partners. In order to demonstrate this approachwe synthesized lactose hydroxylamine 7 by aminooxyacetylation¹⁶ of aglycosylamine derivative. Compound 7 was coupled with the aldehydederived from enzymatic oxidation of 2 to give chemoselectively ligatedglycopeptide 8.

We also applied the enzymatic oxidation and chemoselective ligationreactions to the simple monosaccharide α-benzyl GalNAc to obtainoxime-linked disaccharide 9 for spectroscopic comparison withoxime-linked glycopeptides. Both the trans and cis isomers of thecompound 9 were obtained in a ratio of 2.5:1 (trans/cis).¹⁷ ¹H NMRanalysis of drosocin analog 6, bearing an identical oxime-linkeddisaccharide, revealed only a single isomer which was assigned the transconfiguration based on the chemical shift of the oxime proton (HC═NOR,7.69 ppm). Thus, the C-1 substituent of GalNAc appears to affect thetrans/cis ratio of oximes formed at C-6. Conformational analysis ofoligosaccharides possessing the native GlcNAcβ1ø6GalNAcα1øOR structurehas exposed interactions between the C-1 sustituent of GalNAc and theGlcNAc residue that affect the conformational preference of the β1ø6linkage.¹⁸

Finally, we compared the relative potencies of unglycosylated drosocinand chemoselectively ligated analog 5 (the closest structural mimic tonative GaløGalNAc-drosocin) to determine the functional consequences ofthe unnatural oxime-linked glycan at Thr11. Glycopeptide 5 was found tobe 3 to 4-fold more potent in blocking bacterial growth (IC₅₀=0.12+0.02μM) than unglycosylated drosocin (IC₅₀=0.40±0.05 μM), similar to thetrend observed with native glycosylated drosocin. This observationindicates that flexibility in the structure of the outlying glycoform ispermitted as long as the native sugar-peptide linkage is maintained.Glycoproteins that follow this paradigm are well suited synthetictargets for this chemoselective ligation approach.

General methods: Galactose oxidase (EC 1.1.3.9) (450 units/mg) waspurchased from Sigma. All chemical reagents were obtained fromcommercial suppliers and used without fuirther purification. For flashchromatography, 230-400 mesh silica gel 60 (E. Merck No. 9385) wasemployed. Analytical thin layer chromatography (tlc) was conducted onAnaltech Uniplate silica gel plates with detection by ceric ammoniummolybdate and/or by UV light. Reversed-phase high pressure liquidchromatography (RP-HPLC) was performed on a Rainin Dynamax SD-200 systemusing Microsorb and Dynamax C₁₈ reversed-phase columns (analytical: 4.6mm ID×25 cm, 1 mL/min; semi-preparative: 10 mm ID×25 cm, 3 mL/min), andultraviolet detection (230 nm) was performed with a Rainin Dynamax UV-1detector. The E. coli strain D22 used in bacterial growth inhibitionassays was obtained from the E. coli Genetic Stock Center at YaleUniversity.

Unless otherwise noted, all air and moisture sensitive reactions wereperformed under a nitrogen atmosphere. All solvents were distilled undera nitrogen atmosphere prior to use. THF was dried and deoxygenated overNa/benzophenone; CH₂Cl₂, CH₃CN, and benzene were dried over CaH₂;toluene was dried over Na. Unless otherwise specified, extracts weredried over MgSO₄ and solvents were removed with a rotary vacuumevaporator. The ¹H- and ¹³C-NMR spectra were obtained with BrukerAMX-300 and AMX-400 MHz spectrometers. Chemical shifts are reported in δvalues downfield from tetramethylsilane (TMS) and coupling constants arereported in Hz. Low resolution FAB mass spectra were obtained by theMass Spectrometry Laboratory at the University of California at Berkeleyon a AE 1 M512 mass spectrometer using m-nitrobenzyl alcohol or glycerolas the matrix solvent. Electrospray ionization mass spectrometry(ESI-MS) was performed on a Hewlett-Packard 5989A mass spectrometerequipped with an electrospray ion source.The synthesis of compound 5S is shown in Scheme 1c.

4-O-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl)-2,3,6-tri-O-acetyl-β-D-glucopyranosylazide (1S). To a suspension of 0.033 g (0.147 mmol) of AgClO₄ in 7 mL ofCH₂Cl₂ was added 0.147 mL of a 1 M solution of SnCl₄ in CH₂Cl₂. Thesolution was stirred for 1 h in the dark at rt. A solution of β-lactoseoctaacetate (0.500 g, 0.737 mmol) and azidotrimethylsilane (0.117 mL,0.884 mmol) in CH₂Cl₂ (7 mL) was added to the suspension and thereaction was stirred for 5.5 h. The mixture was diluted with 50 mLCH₂Cl₂ and washed with 25 mL saturated aqueous NaHCO₃ and water (3×15mL). The organic layer was dried and concentrated directly onto silicagel for chromatography. The column was eluted with 2:1 hexanes/ethylacetate to give 0.262 g (54%) of a pale yellow foam; R_(f) 0.54 (1:3hexanes/ethyl acetate); IR (KBr pellet): 3446, 2121,1751,1371, 1232,1059cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.97 (s, 3 H), 2.05 (s, 3 H), 2.06 (s,3 H), 2.07 (s, 3 H), 2.09 (s, 3 H), 2.14 (s, 3 H), 2.15 (s, 3 H), 3.79(m, 3 H), 4.11 (m, 3 H), 4.50 (m, 2 H), 4.86 (t, 1 H, J=8.84), 4.95 (dd,1 H, J=10.45, 3.40), 5.11 (m, 1 H), 5.21 (t, 1 H, J=9.09), 5.35 (d, 1 H,J=2.64); ¹³C NMR (100 MHz, CDCl₃): δ 20.46, 20.60, 20.69, 20.76, 60.76,61.71, 66.57, 69.06, 70.76, 70.91, 72.52, 74.80, 75.76, 77.21, 87.69,101.09, 169.02, 169.44, 169.56, 170.00, 170.06, 170.24, 170.29; FAB-MS:Calcd. for C₂₆H₃₆O₁₇N₃ 662 (M+H), found 662. Anal. Calcd. for C₂₆H₃₅,O₁₇N₃: C, 47.20; H, 5.33; N, 6.35. Found: C, 47.49; H, 5.60; N, 6.31.

4-O-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl)-2,3,6-tri-O-acetyl-β-D-glucopyranosylamine (2S). A solution of 1S (430 mg, 0.65 mmol) and 10% Pd/C (80 mg) inmethanol (20 ml) was stirred under a hydrogen atmosphere for 30 min. Themixture was filtered through Celite and concentrated to give 413 mg(100%) of unstable glycosylamine 2S which was used without furtherpurification in the next reaction.

N^(α)-[4-O-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl]-tert-butoxycarbonylaminooxyacetamide(3S). Crude compound 2S (413, 0.65 mmol) was dissolved in 15 ml offreshly distilled CH₂Cl₂ along with t-butoxycarbonylaminooxy acetic acid(157 mg, 0.80 mmol), 1-hydroxybenzotriazole hydrate (108 mg, 0.80 mmol),and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (153 mg, 0.80 mmol)and stirred at rt for 4.5 h. The reaction solution was diluted with 100mL of CH₂Cl₂ and washed with 0.1M HCl (3×25 mL), water (3×25 mL) andsaturated aqueous NaHCO₃ (3×25 mL). The organic layer was dried andconcentrated directly onto silica gel for chromatography. The column waseluted with 1:1 hexanes/ethyl acetate to give 400 mg (76%) of a whitefoam. ¹H NMR (400 MHz, CDCI₃): δ 1.47 (s, 9 H), 1.96 (s, 3 H), 2.02 (s,3 H), 2.04 (s, 3 H), 2.05 (s, 3 H), 2.06 (s, 3 H), 2.10 (s, 3 H), 2.15(s, 3 H), 3.82 (m, 3H), 4.11 (m, 3H), 4.33 (dd, 2H, J=25.43, 16.41),4.47 (m, 2H), 4.94 (m, 2H), 5.10 (dd, 1H, J=7.78, 10.36), 5.28 (m, 2H),5.34 (d, 1H, J=3.20). ¹³C NMR (100 MHz, CDCl₃): δ 20.50, 20.60, 20.64,20.72, 20.80, 20.85, 28.09, 60.86, 62.08, 66.63, 69.02, 70.73, 71.02,71.10, 72.75, 74.60, 75.97, 77.50, 83.14, 100.95, 157.38, 168.99,169.55, 170.07, 170.15, 170.36, 170.94. FAB-MS: Calcd. for C₃₃H₄₉O₂₁N₂809 (M+H), found 809.

N^(α)-(4-O-β-D-galactopyranosyl)-β-D-glucopyranosyll-tert-butoxycarbonylaminooxyacetamide(4S). To a solution of compound 3S (32 mg, 0.040 mmol) in 3.6 mL MeOHwas added 0.40 mL of NaOMe in MeOH (100 mM). The reaction mixture wasstirred at rt for 7.5 h, neutralized on H⁺ resin and concentrated togive 21 mg (100%) of compound 4S. ¹H NMR (400 MHz, D₂O): δ 3.71 (m, 14H), 4.51 (app d, 3H), 5.06 (d, 1H, J=9.16). ¹³C NMR (100 MHz, D₂O): δ29.94, 30.05, 32.15, 62.37, 63.58, 71.07, 73.47, 74.02, 75.02, 77.16,77.52, 77.88, 79.02, 80.26, 81.38, 105.4, 161.2, 174.9.

N^(α)-[(4-O-β-D-galactopyranosyl)-β-D-glucopyranosyl]-aminooxyacetamide(5S). Compound 4S (21 mg, 0.040 mmol) was dissolved in 3:2 CH₂Cl₂/TFAand stirred at rt for 30 min. The solvent was removed under vacuum andthe residue was coevaporated twice with toluene. The crude product wasdissolved in water, neutralized on anion exchange resin (hydroxide form)and concentrated to afford 17 mg (100%) of free hydroxylamine 5S. ¹H NMR(400 MHz, D₂O): δ 3.73 (m, 14 H), 4.27 (s, 2 H), 4.45 (d, 1H, J=7.40),5.07 (d, 1H, J=9.16). ¹³C NMR(100 MHz, D₂O): δ 62.36, 63.59, 71.10,73.48, 73.94, 75.04, 76.12, 77.54, 77.90, 79.04, 80.26, 81.42, 105.41,176.4. FAB-MS: Calcd. for C₁₄H₂₇O₁₂N₂ (M+H) 415, found 415.

H₂N-Gly-Lys-Pro-Arg-Pro-Tyr-Ser-Pro-Arg-Pro-Thr(α-D-GalNAc)-Ser-His-Pro-Arg-Pro-Ile-Arg-Val-OH(GalNAc-drosocin) (SEQ ID NO:4). The GalNAc-drosocin glycopeptide wassynthesized on HMP resin using N^(α)-Fmoc-protected amino acids andDCC-mediated HOBT ester activation in NMP (ABI 431A synthesizer,user-devised cycles). The glycosylated amino acidN^(α)-Fmoc-Thr(AC3-α-D-GalNAc, 1) (˜2× molar excess) was activated withHBTU in the presence of HOBt and DIEA; acylation was complete within 6h. After completion of peptide synthesis, the sugar moiety wasdeacetylated by treating the resin with 5.5% hydrazine hydrate in MeOHfor 18 h at rt and washing with methanol and diethyl ether. Peptideresin cleavage/deprotection was accomplished with reagent K (4 h, rt)(King, D. S.; Fields, C.; Fields, G. Int. J. Pep. Prot. Res. 1990, 36,225). The purity of the resulting crude glycopeptide was ˜95% asassessed by RP-HPLC and ESI-MS: Calcd. 2401.78 (M⁺), found 2402.13. Thedrosocin glycopeptide was used without further purification insubsequent reactions. The chemical synthesis of unglycosylated drosocinwas accomplished using similar methods.

Galactose oxidase reactions. A solution of GalNAc-drosocin (1 mM) inDulbecco's phosphate buffer saline, pH 7.3, and galactose oxidase (50units) was incubated at 37° C. for 1.5 h. Oxidized GalNAc-drosocin waspurified by semi-preparative RP-HPLC. Elution was accomplished by agradient of acetonitrile in water, both with 0.1% TFA: CH₃CN:H₂O(85:15-70:30) (55 min). The aldehyde product was analyzed by ESI-MS:Calcd. 2399.76, found 2418.29 (M +H₂O, hydrated aldehyde).

Chemoselective ligation reactions. A solution of drosocin glycopeptidealdehyde (0.50 mM) in NaOAc buffer (100 mM, pH 5.5) was incubated withcompound 3, 4 or 7 (final concentration=1.25 mM) at 37° C. for 24 h. Thereactions were monitored by RP-HPLC: elution was accomplished by agradient of acetonitrile in water, both with 0.1% TFA: CH₃CN:H₂O(85:15-70:30) (28min). The respective products 5, 6 and 8 were analyzedby ESI-MS and glycopeptide 6 was further characterized by ¹H NMRanalysis. Glycopeptide 5: Calcd. 2576.92 (M⁺), found 2577.20.Glycopeptide 6: Calcd. 2618.22 (M⁺), found 2617.70. Characteristic peakin the ¹H NMR spectrum (400 MHz, D₂O): δ 7.69 (d, J=3.2) (HC═NOR).Glycopeptide 8: Calcd. 2796.12 (M⁺), found 2796.22.

Bacterial growth inhibition assays. Growth inhibition assays wereperformed essentially as described by Bulet et al. (Eur. J. Biochem.1996, 238, 64). Sterile 96 well plates (Coming #25860) were used, with afinal volume of 40 μl per well. This volume comprised 35 μl of amid-logarithmic phase culture of E. coli D22 at an initial A₅₉₅=0.001 inLuria-Bertani (LB) media containing streptomycin (50 μg/ml), added to 5μl serially diluted peptide (unglycosylated drosocin orGalGalNAc-drosocin) in water. Peptide concentrations were determined bymeasuring dry weight, and assuming 80% of that weight was peptide andthe remainder water and salts. Final concentrations ranged from 10⁻¹¹ to10⁻² M; the precise range used for a given peptide depended on itspotency. Plates were incubated for 6 h at 25° C. with periodic shaking.Growth was determined by measuring the absorbance at 595 nm on a BioRad550 microplate reader. Percent inhibition was defined as((A_(o)−A_(x))/A_(o))*100), where A_(o) was the absorbance of a controlwell lacking peptide and A_(x) was the absorbance of a well withpeptide.

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14. Cao, S.; Tropper, F. D.; Roy, R. Tetrahedron 1995, 51, 6679.

15. Likhosherstov, L. M.; Novikova, O. S.; Derevitskaja, V. A.;Kochetkov, N. K. Carbohydr.Res. 1986,146,C1.

16. Kurth, M.; Pèlegrin, A.; Rose, K.; Offord, R. E.; Pochon, S.; Mach,J. -P.; Buchegger, F. J. Med. Chem. 1993, 36, 1255.

17. The trans and cis isomers of compound 9 were assigned based on thechemical shift of the oxime proton (HC═NOR) in the ¹H NMR spectrum (400MHz, D₂O) (Karabatsos, G. J.; Hsi, N. Tetrahedron 1967, 23, 1079). Transisomer: 7.56 ppm (doublet, J=4.2 Hz); Cis isomer: 6.95 ppm (doublet,J=4.4 Hz).

18. Pollex-Krülger, A.; Meyer, B.; Stuike-Prill, R.; Sinnwell, V.;Matta, K. L.; Brockhausen, I. Glycoconjugate J. 1993,10,365.

All publications and patent applications cited in this specification andall references cited therein are herein incorporated by reference as ifeach individual publication or patent application or reference werespecifically and individually indicated to be incorporated by reference.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A synthetic method comprising the steps of incorporating into apeptide an α-amine protected (2S)-aminolevulinic acid to form aresultant peptide comprising a (2S)-aminolevulinic acid residuecomprising a ketone group; and substituting the ketone group of theresidue with a substituent selected from the group consisting of adetectable label, an O- or N-linked glycosyl group, an aminooxy sugar, ahydrazide sugar, and a thiosemicarbazide-functionalized sugar, whereinthe resultant peptide is incorporated into a cell or cellular structure.2. The method of claim 1, wherein the substituent is a detectable label.3. The method of claim 1, wherein the substituent is a detectable labelcomprising a fluorescent resonance energy transfer (FRET) donor oracceptor.
 4. The method of claim 1, wherein the substituent is an O- orN-linked glycosyl group to yield a glycoconjugate.
 5. The method ofclaim 1, wherein the substituent is an aminooxy sugar to yield acorresponding oxime.
 6. The method of claim 1,wherein the substituent isan aminooxy sugar consisting of aminooxy N-acetyl galactosamine(GalNAc).
 7. The method of claim 1, wherein the substituent is anaminooxy sugar consisting of aminooxy lactose.
 8. The method of claim 1,wherein the substituent is a hydrazide sugar to yield a correspondinghydrazone.
 9. The method of claim 1, wherein the substituent is ahydrazide sugar consisting of lactose succinic hydrazide.
 10. The methodof claim 1, wherein the substituent is athiosemicarbazide-functionalized sugar to yield a correspondingthiosemicarbazone.
 11. The method of claim 1, wherein the substituent isa thiosemicarbazide-functionalized sugar consisting of chitobiosethiosemicarbazide.
 12. The method of claim 1,wherein the substituent isa thiosemicarbazide-functionalized sugar consisting of lactosethiosemicarbazide.
 13. A nonhuman or isolated cell or cellular structureincorporating a peptide comprising a (2S)-aminolevulinic acid residuecomprising a substituted or unsubstituted ketone group, wherein thesubstituted ketone group comprises a substituent selected from the groupconsisting of a detectable label, an O- or N-linked glycosyl group, anaminooxy sugar, a hydrazide sugar, and athiosemicarbazide-finctionalized sugar.